IRAK1 and IRAK4 as emerging therapeutic targets in hematologic malignancies.

PURPOSE OF REVIEW: Cell intrinsic and extrinsic perturbations to inflammatory signaling pathways are a hallmark of development and progression of hematologic malignancies. The interleukin 1 receptor-associated kinases (IRAKs) are a family of related signaling intermediates (IRAK1, IRAK2, IRAK3, IRAK4) that operate at the nexus of multiple inflammatory pathways implicated in the hematologic malignancies. In this review, we explicate the oncogenic role of these kinases and review recent therapeutic advances in the dawning era of IRAK-targeted therapy. RECENT
FINDINGS: Emerging evidence places IRAK signaling at the confluence of adaptive resistance and oncogenesis in the hematologic malignancies and solid tissue tumors. Preclinical investigations nominate the IRAK kinases as targetable molecular dependencies in diverse cancers.
SUMMARY: IRAK-targeted therapies that have matriculated to early phase trials are yielding promising preliminary results. However, studies of IRAK kinase signaling continue to defy conventional signaling models and raise questions as to the design of optimal treatment strategies. Efforts to refine IRAK signaling mechanisms in the malignant context will inspire deliberate IRAK-targeted drug development and informed combination therapy.

INTRODUCTION

The vertebrate innate immune system encompasses a network of phylogenetically conserved molecular and cellular mechanisms that have evolved as an inborn defense against pathogens. A defining feature of the innate immune system, conducted by the toll-like receptors (TLRs), is the ability to recognize generic molecular patterns associated with bacteria, fungi, viruses, and cellular debris. The TLRs, with minor exceptions, converge on the interleukin 1 receptor-associated kinases (IRAK1, IRAK2, IRAK3, IRAK4) to coordinate multiple inflammatory pathways involved in cell survival, cytokine production, and priming of the adaptive immune system. Several recent reviews have meticulously outlined genetic and molecular evidence that cooptation of IRAK-signaling pathways is intrinsic to the pathobiology of the hematologic malignancies [1-9]. These observations have ignited interest in the IRAK kinases as therapeutic targets and thereby renewed focus on resolving IRAK-signaling mechanisms. Accumulating evidence indicates that IRAK signaling is more dynamic and member-specific than what is ascribed by simplistic conventional signaling models. These emerging idiosyncrasies have critical implications for how best to manipulate the IRAK kinases to subvert aberrant inflammatory signaling in malignancy. Below, we provide a contemporary overview of the IRAK kinases in the context of healthy and malignant biology. We profile IRAK-targeted therapies and enumerate essential research mandates for the field.

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CONVENTIONAL SIGNALING PARADIGM OF INTERLEUKIN 1 RECEPTOR-ASSOCIATED KINASES

IRAK4

IRAK4 is a 460 amino acid threonine/serine kinase and the most recent IRAK family member to be identified [10]. Upon ligation, the TLRs and IL-1 Receptor complex (IL-1R1 and IL-1RAcP) dimers undergo a conformational shift that re-orients their cytoplasmic TIR domains [11,12]. Heterotypic IL1R/IL1RAcP and homotypic TLR TIR-TIR dimers (except in the case of TLR3) subsequently recruit the TIR domain-containing adaptor molecule MyD88 [13]. The N-terminal death domains of MyD88, IRAK4, and IRAK2 then facilitate the assembly of a multimeric, helical signaling complex (Myddosome) consisting of six molecules of MyD88, four molecules of IRAK4, and four molecules of IRAK2 [14,15]. IRAK4 dimers recruited to the Myddosome undergo asymmetric trans-autophosphorylation [16] and recruit IRAK1, which then itself participates in extensive autophosphorylation and dissociates from the Myddosome (Fig. 1) [17]. Intriguingly, the absolute requirement of IRAK4 kinase activity for canonical signaling is not firmly established, and few IRAK4 substrates have been characterized. Vollmer et al.[18] found that chemical inhibition of IRAK4 autophosphorylation in IL-1R cells had a negligible effect on IRAK1 activity and NF-κB/P38/JNK signaling upon IL-1 stimulation. Others have similarly demonstrated that expression of kinase-dead IRAK4 isoforms in IRAK4-deficient cells fully restores IRAK1 phosphorylation and signaling downstream of IL-1 [19,20]. In an IL-1-stimulated fibroblast model, Ferrao et al.[16] found that ablation of catalytic activity or dimerization only moderately impaired the ability of IRAK isoforms to conduct signaling and induce downstream cytokine expression. Conversely, Wang et al.[21] found that only the phosphorylated species of IRAK4 was capable of heterodimerizing with IRAK1. In the human monocytic acute myeloid leukemia (AML) THP-1 cell line, an IRAK4 kinase inhibitor demonstrated an equivalent ability to impair TLR4-induced signaling through the NF-κB, JNK, and MAPK pathways as an IRAK4 PROTAC built on the same compound, suggesting that IRAK4 kinase activity does, in fact, mediate signaling [22]. However, both the IRAK4 inhibitor and proteolysis-targeting chimera (PROTAC) degrader only modestly attenuated canonical signaling, calling into question whether Myddosome assembly entirely requires IRAK4 in any capacity [22]. Collectively, though some studies report a requisite role of IRAK4 catalytic activity for signal transduction and cytokine induction in human myeloid cells [10,23,24], the majority of experimental evidence insinuating an absolute requirement of IRAK4 catalytic activity in canonical signaling comes from mouse models. This is consistent with the published finding that murine myeloid cells are far more dependent on IRAK4 for inflammatory signaling than human myeloid cells [25]. Thus, the requirement of IRAK4 kinase activity may vary by cell type, upstream stimulus, and pathological context.

FIGURE 1

Dysregulation of interleukin 1 receptor-associated kinase-dependent signaling in myeloid malignancies. Inflammatory and immune-related receptors, such as certain TLRs and IL-1R, recruit the adaptors TIRAP and MyD88, along with IRAK kinases and TRAF6, to form the Myddosome complex. Negative regulators of the pathways, such as miR-145, miR-146a, and TIFAB, are frequently deleted in MDS and AML. Mutations in U2AF1 and SF3B1 result in conversion of hypomorphic (IRAK4-S) to hypermorphic (IRAK4-L) IRAK4 isoforms in MDS and AML. IRAK4-L can recruit MyD88 and IRAK1 to activate signaling to NF-κB and MAPKs. The signaling pathway was adapted from Trowbridge and Starczynowski (2021). AML, acute myeloid leukemia; IRAK, interleukin 1 receptor-associated kinases; MDS, myelodysplastic syndromes; TLRs, toll-like receptors.

IRAK1

IRAK1 is a threonine/serine kinase. Unlike IRAK4, IRAK1 is not a core constituent of the Myddosome but transiently interacts with IRAK4 upon Myddosome assembly. Unlike IRAK4, IRAK1 exists primarily in an inactive conformation at the basal state and requires upstream stimulation for rapid activation [18]. IRAK1 is reported to be a phosphorylation substrate of IRAK4 [10] and is subject to extensive ubiquitylation and autophosphorylation proceeding IL-1R/TLR stimulation [17,26]. However, IRAK4-mediated activation of IRAK1 kinase activity occurs by an allosteric mechanism rather than by covalent modification [18]. Once dissociated from the Myddosome, IRAK1 interacts with and activates the E3 ligase TRAF6 to induce canonical signaling through the NF-κB and MAPK pathways [27] (Fig. 1). Intriguingly, lysine (K) 63-linked polyubiquitylation of IRAK1 is necessary for TRAF6-mediated signal transduction, suggesting that covalent modification of IRAK1 may regulate its accessibility as a scaffolding substrate [17,28]. Precisely how IRAK1 stimulates TRAF6 remains obscure, though it is apparently independent of IRAK1 catalytic activity; thus, in at least some contexts, the canonical IRAK signaling axes are robust to the isolated loss of either IRAK1 or IRAK4 kinase activity. It is tempting to speculate that IRAK1 and IRAK4 maintain catalytic redundancy in this regard, therefore, requiring a bispecific approach to terminate canonical signaling by small molecule inhibition. We discuss preliminary efforts to target the IRAK kinases by such an approach below.

Recent efforts have focused on the role of IRAK1 in activating NLRP3 inflammasome assembly. Several groups have reported that IRAK1 regulates rapid NLRP3 inflammasome assembly and caspase 1 cleavage following IL-1R/TLR ligation in a transcription-independent fashion [29,30]. Notably, and in contrast to Myddosome signaling via TRAF6, NLRP3 inflammasome assembly is dependent on the catalytic competency of both IRAK1 and IRAK4 [29]. These findings contradict to a study that asserts a role for IRAK1 as a repressor of NLRP3 inflammasome assembly [31]. The IRAK1 substrates and interactors in this context are not characterized, and temporally segregated interactions with distinct binding partners may modulate the influence of IRAK1 on inflammasome assembly.

IRAK2

IRAK2, like IRAK4, is a core constituent of the Myddosome complex. Unlike IRAK1 and IRAK4, IRAK2 is predicted to be a pseudokinase as it lacks a critical aspartate residue in the VIb kinase subdomain [32] and exhibits negligible kinase activity in vitro[33]. However, the fundamental signaling mechanisms by which IRAK2 operates have not been subject to the same level of scientific scrutiny as have IRAK1 and IRAK4, and evidence from human models is conspicuously absent. This is especially problematic as the utilization of IRAK2 in mice and humans is discrepant [25]. As with IRAK1, IRAK2 contains TRAF6-binding sites [34], and IRAK2 overexpression is sufficient to induce TRAF6 ubiquitylation and NF-κB signaling in human cell lines [35,36]. Moreover, knockdown of IRAK2 in human cell lines suppresses NF-κB signaling in response to TLR stimulation [36]. Several investigations in mouse models posit that IRAK2 is indeed catalytically active and maintains late-phase NF-κB signaling and sustained cytokine production downstream of TLR stimulation [37-40]. IRAK2 has not been convincingly demonstrated to possess such catalytic competence and latent signaling regulation in human biology.

IRAK3

IRAK3, also known as IRAK-M to reflect its predominant expression in monocytes, is also a pseudokinase [33]. In contrast to the other IRAK family members, studies in human models indicate IRAK3 is a negative regulator of Myddosome signaling [41-44], which supports the association of inactivating IRAK-M mutations with early-onset asthma [45]. Like all family members, IRAK3 possesses an N-terminal death domain, and as with IRAK1 and IRAK2, a C-terminal domain with TRAF6 binding sites [34]. IRAK3 is, thus equipped to engage the Myddosome complex and modulate TRAF6, though the exact mechanism by which IRAK3 suppresses Myddosome signaling is obscure. Crystal structure analysis of the IRAK3 pseudokinase domain predicts a higher-order assembly of IRAK3 dimers and IRAK4 that would occlude the IRAK4 catalytic site, presenting a plausible means of mitigating Myddosome signaling [46]. Along these lines, mutagenesis of N-terminal IRAK3 residues predicted to mediate interaction with IRAK4 abrogate the ability of IRAK3 to dampen TLR signaling [42]. These interactions with IRAK4 may serve to prevent Myddosome disassembly and TRAF6 activation, as observed previously [41]. IRAK3 also possesses intrinsic guanylate cyclase activity central to its regulatory competence, possibly by creating a localized cGMP gradient that stabilizes the Myddosome [43]. Finally, an alternative model posits that an IRAK3-integrated Myddosome complex engages a MEKK3-dependent NF-κB pathway that drives the expression of anti-inflammatory genes [47].

ROLE OF INTERLEUKIN 1 RECEPTOR-ASSOCIATED KINASES IN HEMATOLOGIC MALIGNANCIES

Inflammatory dysregulation is a fundamental feature of pathogenesis in the hematologic malignancies. Multiple oncogenic signaling nodes converge on the IRAK kinases, generating significant interest in these kinases as targetable effectors of malignancy. In this section, we discuss accumulated evidence pertaining to the role of the IRAK kinases in the hematologic malignancies.

Myelodysplastic syndromes

The myelodysplastic syndromes (MDS) are a group of diseases characterized by ineffective hematopoiesis and myelodysplasia driven by the clonal dominance of defective hematopoietic stem cells (HSC) [48]. Many of the genetic lesions associated with MDS induce cell-intrinsic alterations in the IRAK-signaling hub [49-54]. One of the most common subtypes of MDS, 5q-syndrome, is mediated by the deletion of microRNAs (miR-145 and miR-146a) and regulatory proteins (TIFAB) that target IRAK1 and TRAF6 (Fig. 1). Haploinsufficiency of these genes depresses TRAF6 and IRAK1 and promotes NF-κB-dependent myeloproliferation [51,52,55-58]. TLR1, TLR2, TLR4, TLR6, MYD88, and IRAK1 are frequently overexpressed in MDS HSC, supporting a functional role for IRAK signaling in MDS pathobiology [59-63]. Rhyasen et al.[64] reported that inhibition of IRAK1 reduced progenitor function and viability in MDS HSC but not healthy CD34+ bone marrow cells. Driver mutations in the splicing factors U2AF1 and SF3B1 that induce hypermorphic IRAK4 isoforms are recurrent in MDS and AML and impose sensitivity to IRAK4 inhibition [65,66]. Stromal elaboration of the alarmins S100A8 and S100A9, which ligate TLR4, drives genotoxic stress and preleukemic evolution, suggesting the IRAK-signaling axis may play a pivotal role in the emergence of MDS HSC [67]. Indeed, the alarmins are noted to be elevated in the plasma of low-risk and intermediate-risk MDS [68] and proposed to serve a multifaceted role in disease progression by enforcing an erythroid differentiation block, driving myeloid-derived suppressor cell (MDSC) expansion, and instigating NRLP3 inflammasome activation [50,69-71,72]. S100A9 binding to the CD33 complex on MDSC promotes MDSC outgrowth and the production of cytokines that suppress hematopoiesis and antitumor immunity [71] MDSC are overrepresented in the bone marrow of MDS, and their abundance is a negative prognostic factor [73]. MDSCs themselves produce S100A8/9, instituting a forward TLR signaling feedback loop [74]. The S100A8/9 alarmins also enforce hematopoietic dysfunction in MDS by triggering NLRP3 inflammasome assembly and pyroptotic cell death in MDS HSCs via TLR4 signaling [72]. As discussed above, IRAK1 is a critical mediator of NLRP3 inflammasome assembly downstream of the TLRs [29,30]. Inflammasome priming also requires TLR-IRAK-mediated activation of NF-κB to induce NRLP3 and IL-1β expression [75]. IRAK1 thereby plays a dual role in regulating the NLRP3 inflammasome at the transcriptional and protein level. Furthermore, the NLRP3 inflammasome facilitates the maturation and release of IL-1 β, a cytokine that signals through the IRAK kinases. Overexpression of IL1RAcP is another common feature in MDS HSC, and IL-1 signaling drives the selective expansion of malignant myeloid clones [76]. This is consistent with the recent finding that chronic exposure to the TLR ligand lipopolysaccharide (LPS) elicits a competitive advantage of MDS HSCs in chimeric bone marrow mouse models [77]. Collectively, these findings suggest that IRAK signaling downstream of the IL-1 receptor or TLR family favors the dominance of MDS clones. Thus, the IRAK signaling complex resides at multiple nodes in a complex inflammatory circuit governing MDS and represents a logical target to subvert various disease mechanisms concurrently.

Acute myeloid leukemia

MDS often antecedes AML, and many of the IRAK-signaling dependencies in MDS are preserved in AML. In an induced pluripotent stem cell (iPSC) model that harnessed sequential CRISPR/Cas9 mutagenesis to recapitulate clonal progression to AML, Wang et al. integrated transcriptional and epigenetic analyses to consolidate genetic dependencies in the transformation to overt leukemia. These dependent genes were heavily enriched for inflammatory mediators, and an IRAK1/IRAK4 dual inhibitor subverted progenitor function in leukemic, but not parental, iPSC-derived hematopoietic cells [78]. These findings suggest that signaling through the IRAK kinases is recruited early in the transition to AML. Mutually exclusive expression of a hypermorphic long IRAK4 (IRAK4-L) isoform over a hypomorphic short (IRAK4-S) isoform predicts clinical aggressiveness and worse outcomes in AML patients [65]. Treatment with an IRAK4 inhibitor potently suppresses leukemic function in AML cell lines with predominant IRAK4-L expression [65]. In concordance, parallel inhibition of the JNK and NF-κB pathways, which are both regulated by the IRAK kinases, effectively eradicates AML stem and progenitor cells [79]. As mentioned previously, IL-1 signaling favors the competitive advantage of leukemic clones in MDS and AML [76]. IRAK1 hyperphosphorylation is also observed in AML of heterogeneous genetic backgrounds, and IRAK1 inhibition reduces viability and in vivo leukemic burden across diverse AML cell lines and primary patient samples [80]. Nevertheless, the precise role of IRAK1/4 signaling dependencies in AML remains unknown. Whether the efficacy of IRAK1 inhibition and IRAK4 inhibition is mediated by repression of mutual or divergent signaling pathways, or, in the latter case, whether functional redundancy permits reciprocal compensation is not clear. These questions are of considerable clinical interest, as the attainable development of dual selective IRAK1/4 inhibitors may offer substantial therapeutic benefit over extant compounds. Furthermore, the requirement of upstream receptor ligation to prime the oncogenic function of the IRAK kinases in AML is obscure. These questions must be a priority for future investigations.

Other leukemias

IRAK4 has emerged as a target in chronic lymphocytic leukemia, which occasionally harbors driver mutations in MYD88 and TLR receptors [81-83]. MYD88-mutated CLL cases manifest an NF-κB signature, though treatment with an experimental IRAK4 inhibitor reduces viability and proliferation in patient-derived CLL regardless of MYD88 status [84,85]. IRAK1 is overexpressed in multiple T-ALL subtypes, which undergo apoptosis and cell-cycle disruption upon IRAK1 knockdown [86]. Notably, treatment with an IRAK inhibitor in this study only partially recapitulated the efficacy of protein knockdown, hinting that kinase-independent IRAK functions are indeed relevant to these malignancies [86]. A polymorphism that is predicted to mitigate the ability of miR146a – one of the microRNAs deleted in del(5q) MDS – to repress IRAK1 and TRAF6 is associated with an increased incidence of childhood ALL in a Taiwanese cohort [87]. TLR activation is observed in lymph node-resident CLL cells, and combination therapy of an IRAK1/4 inhibitor with the BTK inhibitor Ibrutinib demonstrated superiority over single-agent therapy in a preclinical study [88]. IRAK3, a suppressor of the IRAK1/2/4-signaling complex, is robustly expressed in monocytes exposed to circulating leukemic cells in CML patients via engagement of TLR4 and CD44 [89]. IRAK3 induction is theorized to deactivate monocytes and enable immune tolerance in CML [89]. Therefore, targeting IRAK3 in myeloid cells may facilitate an antitumor immune response in CML and possibly other heme malignancies.

Lymphomas

Activating MYD88 mutations that promote tonic signaling through the MyD88-IRAK signaling complex are found in nearly all cases of Waldenstrom's macroglobulinemia and in a substantial portion of the Activated B-cell (ABC) subtype of Diffuse Large B-Cell Lymphoma (DLBCL) [90-92]. Primary lymphoplasmacytic cells collected from the bone marrow of MYD88-mutant Waldenstrom's macroglobulinemia patients on sustained ibrutinib therapy demonstrate elevated IRAK1 and IRAK4 phosphorylation and synergistic sensitivity to combination therapy of ibrutinib with an IRAK1/4 inhibitor in vitro[93]. An RNAi screen and treatment with an IRAK1/4 inhibitor identified both IRAK1 and IRAK4 as molecular dependencies in MYD88-mutant ABC DLBCL [92]. Recently, Hatcher et al.[94] discovered a highly selective inhibitor of IRAK1 that displays antiproliferative potency in MYD88 mutant lymphoma.

Likewise, a Pyrrolopyrimidine compound with optimized IRAK4 selectively has been developed and found to reduce NF-κB signaling and MYD88-mutant lymphoma viability [95]. It is worth noting that several studies in MYD88 mutant lymphomas describe a predominant reliance on IRAK1 over IRAK4 [22,93,96], and genome-wide CRISPR screens have implicated IRAK1, but not IRAK4, as a significant molecular dependency in ABC DLBCL [97,98]. Indeed, one study reported that IRAK4 was entirely dispensable to ABC DLBCL survival [22]. These data challenge the dogma that IRAK1 absolutely requires upstream recruitment by IRAK4 to initiate signaling, at least in the context of malignant biology. Rather, IRAK4 and IRAK2 may be interchangeable in the mutant Myddosome, or IRAK1 may assert additional Myddosome-independent oncogenic function. Such possibilities should be explored in future studies. Given the equivocal requirement of IRAK kinase activity in Myddosome signaling as alluded to previously, it is reasonable to question whether ATP-competitive small molecule inhibitors are ideal modalities for IRAK-directed therapy. In fact, the studies demonstrating the utility of IRAK1/4 inhibitors in MYD88 mutant lymphoma only observed efficacy in the micromolar ranges, far above the respectively reported IC50 values [91,92,94,95]. In one study of ABC DLBCL, kinase-dead IRAK1 was able to rescue viability following IRAK1 knockdown [92]. Conversely, a recently generated series of IRAK1 PROTACS exhibited antiproliferative potency against ABC DLBCL in the low nanomolar range [99]. These findings indicate the scaffolding functions of IRAK1 facilitate the oncogenic signaling in MYD88-mutant ABC DLBCL.

The IRAK kinases are also implicated in other B-cell malignancies. Genome-wide expression analysis identified IRAK1 expression as one of the two most powerful transcriptomic predictors of follicular lymphoma transformation to more aggressive disease [100]. Nonsynonymous IRAK1 polymorphisms that confer gain-of-function were identified in all cases of a cohort of Kaposi sarcoma-associated herpesvirus-positive primary effusion lymphoma patients where IRAK1 expression was required for tumor growth in culture [101]. TLR stimulation promotes proliferation in splenic marginal zone lymphoma samples in an IRAK1/4-dependent fashion [102]. TLR4 ligation similarly augments proliferation and therapy evasion in a multiple myeloma model [103]. A phase 2 trial reported that the IL-1 receptor antagonist Anakinra reduced proliferation and delayed progression to active disease by reducing downstream IL-6 induction in a subset of patients with smoldering or indolent myeloma [104].

ROLE OF INTERLEUKIN 1 RECEPTOR-ASSOCIATED KINASES IN SOLID TISSUE TUMORS

By and large, IRAK signaling has been studied in the context of normal and malignant leukocyte biology. However, an increasing body of evidence implicates IRAK kinase signaling as a critical effector of tumorigenesis in solid tissue cancers. IRAK4 signaling is a determinant of chemoresistance and disease progression in pancreatic and colorectal cancer [105,106], and IRAK4 expression is proposed to be a biomarker and predictor of poor prognosis in IDH wild-type and 1p19p nonco-deletion gliomas [107]. IRAK1 is purported to be a driver of oncogenesis or chemoresistance in head and neck cancers [108-110], breast cancer [111,112], hepatocellular carcinoma [113-115], nonsmall cell lung cancer [116,117], ovarian cancer [118], cervical cancer [119], melanoma [120], and gastric cancer [121]. IRAK1 is also implicated in supporting tumor-intrinsic resistance to radiation therapy [122] and IRAK1/4 inhibition has demonstrated adjunctive therapeutic benefit in preclinical models of anaplastic thyroid cancer and melanoma [123,124]. TLR9 overexpression is observed in prostate cancer, lending indirect support to the involvement in IRAK signaling in this malignancy as well [125]. A detailed dissection of IRAK signaling across solid tissue cancer is beyond the scope of this review; however, it is worth discussing several cases that highlight the oncogenic roles of IRAK2 and IRAK3. Evaluation of IRAK2 and IRAK3 is conspicuously absent in the hematologic malignancy arena, though the functions outlined below may be conserved features of malignant biology. IRAK3 is primarily recognized as a negative regulator of canonical Myddosome signaling and a mediator immunosuppressive cytokine production [41,47], and is being examined as an effector of tumorigenic immune evasion. Human lung cancer lines induce IRAK3 expression in co-cultured monocytes, and IRAK3 deletion significantly reduces implanted tumor growth in mice, consistent with a role for IRAK3 in mollifying the antitumor response of tumor-associated macrophages [126]. This finding bolsters the observation that leukemic cells in CML induce IRAK3 expression in CD14+ monocytes [89]. It would be worthwhile to assess the existence of such a phenomenon in AML and other leukemias that are notoriously refractory to conventional immunotherapies. Tumor-intrinsic epigenetic repression of IRAK3 is common in hepatocellular carcinoma and it is associated with a less favorable prognosis [127]. These findings indicate that IRAK3 may serve as a tumor suppressor by regulating signaling through the other IRAK family members.

IRAK2 is thought to be redundant with IRAK1 in mice, though the role of IRAK2 in human biology is less certain. It is nonetheless surprising that many reports advocate a tumor suppressor functionality for IRAK2. An IRAK2 SNP that promotes IRAK2 expression is associated with increased overall survival in NSCLC [128], whereas a nonsynonymous SNP that produces a hypofunctional IRAK2 isoform is associated with increased risk of death by colorectal cancer [129]. One study proposes that IRAK2 mitigates oncogenesis in colon cancer by phosphorylating the E3 ligase Smurf1 to trigger self-degradation [130]. Yu et al.[131] discovered that IRAK2 expression sensitizes oral squamous cell carcinoma to radiotherapy by instigating FADD (Fas-associated protein with death domain), an adaptor molecule involved in apoptotic signaling-driven apoptosis.

Notably, FADD contains a death domain (a uniting feature of Myddosome constituents), inviting the possibility that IRAK2 and other IRAK kinases participate in regulatory interactions with FADD. These models conflict with the notion that IRAK2 is a pseudokinase with activity that is confined to its constituency in the Myddosome and warrant further investigation of IRAK2 in the heme malignancies.

THERAPEUTIC APPROACHES TARGETING INTERLEUKIN 1 RECEPTOR-ASSOCIATED KINASES IN HUMAN CANCERS

Several parallel efforts to develop IRAK-targeted therapies for the hematological malignancies are ongoing (Table 1). Yet, IRAK inhibition as a treatment modality for these diseases is still a nascent concept. Several critical questions will need to be addressed before IRAK-targeted therapies achieve widespread clinical use. In this section, we address significant knowledge gaps, explicate the potential use of IRAK-targeted therapies in clinical practice, and highlight preliminary clinical trials.

Table 1

Preclinical and clinical interleukin 1 receptor-associated kinase inhibitors

Compound	Source	Target	Mechanism	Disease Applications	Status
‘23’	AstraZeneca	IRAK3 (DC50 = 2 nmol/l)	PROTAC	Undetermined	Preclinical
		IRAK4 (IC50 = 5 nmol/l)
’AZ1495’	AstraZeneca	IRAK1 (IC50 = 24 nmol/l)	Kinase inhibitor	Non-Hodgkin's lymphoma	Preclinical
BAY1830839	Bayer	IRAK4 (IC50 = 3 nmol/l)	Kinase inhibitor	Rheumatoid arthritis	Clinical
‘Compound 9’	GlaxoSmithKline	IRAK4 (DC50 = 36 nmol/l)	PROTAC	Undetermined	Preclinical
				Non-Hodgkin's lymphoma
		IRAK4 (IC50 = 50 nmol/l)		Waldenstrom's macroglobulenemia
Emavusertib (CA-4948)	Curis	FLT3	Kinase inhibitor	Acute myelogenous Leukemia	Clinical
				Myelodysplastic syndrome
				Chronic lymphocytic Leukemia
HS-243	Duke University	IRAK1 (IC50 = 24 nmol/l)	Kinase inhibitor	Undetermined	Preclinical
		IRAK4 (IC50 = 200 nmol/l)
IRAK-1–4 Inhibitor I	Amgen	IRAK1 (IC50 = 300 nmol/l)	Kinase inhibitor	Not applicable	Preclinical
JH-X-119-01	Dana Farber Cancer Institute	IRAK1 (IC50 = 9 nmol/l)	Kinase inhibitor	Non-Hodgkin's lymphoma	Preclinical
JNJ-1013	Janssen Pharmaceuticals	IRAK1 (DC50 = 3 nmol/l)	PROTAC	Non-Hodgkin's lymphoma	Preclinical
				Hidradenitis suppurtiva
KT-474	Kymera Therapeutics	IRAK4 (DC50 = 2.1 nmol/l)	PROTAC	Atopic dermatitis	Clinical
				Non-Hodgkin's lymphoma
		IRAK4 (IC50 = 0.8 nmol/l)
NCGC1481	Kurome Therapeutics	IRAK1 (IC50 = 22.6 nmol/l)	Kinase inhibitor	Myelodysplastic syndrome	Preclinical
		FLT3 (IC50 < 0.5 nmol/l)		Acute myelogenous leukemia
		JAK2 (IC50 = 23 nmol/l)
Pacritinib	CTI Biopharma	FLT3 (IC50 = 22 nmol/l)	Kinase inhibitor	Myeloproliferative neoplasms	Clinical
		IRAK1 (IC50 < 20 nmol/l)
		IRAK4		Myelodysplastic syndrome
R289	Rigel Pharmaceuticals	IRAK1 IRAK4	Kinase inhibitor	Rheumatological diseases	Clinical
Zabedosertib (BAY1834845)	Bayer	IRAK4 (IC50 = 3.4 nmol/l)	Kinase inhibitor	Rheumatological diseases	Clinical
Zimlovisertib (PF-06650833)	Pfizer	IRAK4 (IC50 = 0.2 nmol/l)	Kinase inhibitor	Rheumatoid arthritis	Clinical

Interleukin 1 receptor-associated kinase inhibitors as monotherapies

One of the major questions concerning IRAK-targeted therapies is whether they will demonstrate efficacy as monotherapies, and, if not, what an informed combination strategy would entail. Phase 1 trials with the compound CA-4948, a small molecule inhibitor with affinity for IRAK4 and FLT3 (Curis Inc), provide the best available data on this question. A phase 1 monotherapy trial in refractory non-Hodgkin's lymphoma (NCT03328078) reported a favorable safety profile and pharmacokinetic properties, with 8 of 28 patients experiencing a reduction in tumor burden of at least 20% [132]. An interim report from a phase 1 open-label trial (NCT04278768) using the same compound as a monotherapy in adults with AML or high-risk MDS described a reduction in bone marrow blasts in 10 of 12 patients who presented with elevated blast counts. Intriguingly, three of the four patients who achieved a complete response had mutations in the U2AF1 or SF3B1-splicing factors, which are known to drive disease by inducing hypermorphic IRAK4 isoforms [65,66]. These encouraging preliminary results bode well for the potential use of IRAK-targeted monotherapy in patients with splicing factor mutations and suggest efficacy in other genetic backgrounds. However, complementation with other drugs may be required for optimum effect in nonsplicing factor mutant AML/MDS. Kymera, operating under the logic that both IRAK4 loss-of-function and immunomodulatory imide drugs (IMiD) suppress NF-κB, have developed first-of-class ‘IRAKIMiD’ PROTACs. IRAKIMiDs consist of IRAK4 ligands coupled to an IMiD (lenalidomide or pomalidomide), thereby recruiting the E3 ligase cereblon to IRAK4 and the IMiD substrate transcription factors Ikaros and Aiolos to catalyze their ubiquitylation and proteasomal degradation. A candidate IRAKIMiD has demonstrated promise in preclinical MYD88-mutant lymphoma models with in-human trials planned for 2021 [133]. The absence of IRAK1-selective drugs in clinical trials makes it difficult to extrapolate the suitability of IRAK1 as a target for monotherapy or compare the relative utility of targeting IRAK1 over IRAK4. Pacritinib, a multikinase inhibitor of JAK2, FLT3, and IRAK1 developed as a treatment for myelofibrosis, demonstrated efficacy in a pilot phase 1 trial of FLT3-ITD AML [134]. Given the promiscuity of this compound, it is impossible to disentangle the individual contributions of IRAK1 and FLT3 inhibition to the observed response. However, one preclinical study reported that pacritinib exhibited potency against primary AML samples of diverse genetic backgrounds, whereas the efficacy of a FLT3 inhibitor lacking affinity for IRAK1 was restricted to samples harboring FLT3-ITD [80]. This study concords with the results of a phase 3 trial in myelofibrosis that identified a response to pacritinib regardless of JAK2V617F allele burden [135]. Further, recently generated IRAK1 inhibitors and PROTACS are effective in constraining MYD88 mutant lymphomas in vitro[94,99]. These results establish a precedent for the clinical development of IRAK1 inhibitors and intimate that pacritinib, by virtue of its activity against IRAK, may be a useful drug in the hematologic malignancies regardless of mutational background.

The matter of which drugs to pair with IRAK-targeted therapy captures two related issues; first, the resistance mechanisms to IRAK-targeted therapies are largely unknown, and; second that recruitment of IRAK signaling may underly adaptive resistance to other therapies. Parallel IRAK signaling and BCR-BTK signaling converge on NF-κB in B-cell lymphomas [92,136], justifying the combination of IRAK-targeted therapies with a BTK inhibitor. The synergy of IRAK inhibitors with the BTK inhibitor ibrutinib affirmed in multiple preclinical studies [93-95,137,138], and NCT03328078 has expanded to enroll refractory lymphoma patients for dual therapy with CA-4948 and ibrutinib. Rhyasen et al.[64] identified upregulation of the antiapoptotic factor BCL2 in MDS/AML clones that escaped IRAK1 inhibitor treatment, with a synergistic reduction of cell expansion and viability obtained upon the addition of BCL2 inhibitors. Similarly, BCL2 inhibitor supplementation dramatically augmented a meager response to single-agent IRAK1/4 inhibitor treatment in a xenograft model of T-ALL [139]. This result invites a potential synergistic combination of IRAK-targeted therapies with the BCL2 inhibitor venetoclax. Indeed, the trial with CA-4948 in AML and high-risk AML (NCT04278768) now includes a venetoclax dual therapy arm. Preliminary results from this arm have not been released at the time of publication. BCL2 upregulation remains the only described adaptation to IRAK inhibitor treatment in AML/MDS; a thorough delineation of escape mechanisms is an eminent need for future studies.

Interleukin 1 receptor-associated kinase inhibitors to overcome adaptive resistance mechanisms to therapy

An expanding body of evidence implicates IRAK signaling as a mechanism of adaptive resistance to various forms of cancer therapy, indicating that IRAK-targeted drugs may realize widespread application as adjunctive therapies. IRAK signaling promotes chemoresistance in diverse preclinical solid-tissue cancer models in which IRAK inhibition restores sensitivity to SN-38, 5-FU, oxaliplatin, gemcitabine, paclitaxel, sorafenib, doxorubicin, and the irinotecan metabolite SN-P38 [105,106,109,112,113,140,141]. Recent investigations assert IRAK signaling as a mechanism of adaptive resistance in the hematologic malignancies as well. Melgar et al.[142] performed kinome screening and gene expression analysis in FLT3-ITD mutant AML cell lines treated with clinical FLT3 inhibitors and revealed activation of IRAK-mediated innate immune signaling to be a mechanism of acute adaptation to FLT3 inhibition. A novel IRAK1/IRAK4/FLT3 tri-selective inhibitor eradicated adaptively resistant AML clones in liquid culture and xenografts [142,143]. Two subsequent studies reiterate innate immune activation [144] and upregulation of the TLR ligand S100A9 [145], respectively, as mediators of adaptive response to the FLT3 inhibition in AML. Du et al.[146] report that treatment with Ara-C or anthracyclines induces a subpopulation of leukemic cells to acquire an inflammatory signature and senescence-like state that enables chemoresistance and disease recurrence. Although the IRAK kinases were not explicitly evaluated, the inflammatory signatures in the resistant leukemic population are enriched for pathways regulated by the IRAK kinases.

No inhibitors or degraders of IRAK2 or IRAK3 are currently being vetted for therapeutic application, though an IRAK3-selective PROTAC has recently been developed [147]. It will be fascinating to see whether targeting IRAK3 demonstrates broad utility in stimulating antitumor immune responses, possibly as part of a combination with checkpoint inhibitors as proposed elsewhere [148].

CONCLUSION

An extensive body of literature implicating IRAK signaling in the hematologic malignancies and promising preliminary clinical trial data validate IRAK-targeted therapy as an exciting approach to the hematologic malignancies. However, these therapies were constructed on an incomplete model of IRAK signaling in cancer. The data reviewed above conflict with the convention that IRAK4 and IRAK1 signal in a linear and interdependent fashion. Moreover, the current conception of IRAK2-signaling functions is problematically gleaned from mouse models, and IRAK2 functionality in the human system is nebulous. These conspicuous gaps in the literature cast doubt as to whether extant IRAK inhibitors are fully optimized for clinical use. Future investigations must address the contribution of IRAK1/IRAK4 scaffolding functions, refine the role of IRAK2 in human malignancy, and evaluate the capacity for signaling redundancy among IRAK family members. These studies are direly needed to settle debate concerning the prioritization of IRAK degraders or ATP-competitive inhibitors, inform ideal target selection, and resolve the demand for a multikinase approach, respectively.

Acknowledgements

We would like to thank members of the Starczynowski labs for helpful suggestions and editing of the review. Figures and illustrations were inspired by BioRender.com.

Financial support and sponsorship

D.T.S. is supported by Cincinnati Children's Hospital Research Foundation, Leukemia Lymphoma Society, National Institute of Health (R35HL135787, R01DK102759, R01DK113639), The Edward P Evans Foundation, and Cancer Free Kids grants. J.B. is supported by National Institutes of Health T32CA117846-04.

Conflicts of interest

D.T.S. serves on the scientific advisory board at Kurome Therapeutics, and is a consultant for Kymera Therapeutics, Kurome Therapeutics, Captor Therapeutics, and Tolero Therapeutics. D.T.S. has equity in Kurome Therapeutics. The other authors declare no competing financial interests.